SIGNIFICANCE: G protein-coupled receptors (GPCRs) activate heterotrimeric G proteins, which together form one of the most important signaling axes found in the cell. Because GPCRs are very common targets for therapeutic drugs, the mechanisms that underlie their regulation are of high biomedical importance. Although it is known that many cytoplasmic factors regulate the activity of G proteins after GPCR-mediated activation, they remain greatly understudied as an untapped opportunity for therapeutic intervention. My goal here is to characterize a novel cytoplasmic regulator of G proteins that operates through modulation of neurotransmission, and has been shown to be relevant in the context of neurological disorders including chronic pain and epilepsy. Current treatments for these diseases include addictive opioids in the case of pain, or a trial-and-error drug seeking process for epilepsy that still leaves approximately 1/3 of patients with ineffective treatments. For this reason, in this proposal I will study the molecular mechanism by how this novel regulator controls GPCR-G protein neuronal signaling. Elucidating the molecular mechanisms of this physiologically important G protein regulator is a first step towards the development of novel targeted treatments for diseases that arise from dysregulated GPCR signaling. BACKGROUND: In the course of a screen for G protein activators that bind to G?i subunits, my Sponsor's laboratory identified a protein that regulates G proteins via a unique and novel mechanism. We coined the term ?paradoxical G protein regulator? (PGR) to convey that it upregulates the modulation of some G protein effectors while simultaneously downregulating the modulation of other G protein effectors. Others had found that loss of this ?PGR? alters GPCR signaling in neurons of the peripheral nervous system and causes chronic pain. More recently my Sponsor's laboratory has found that PGR KO mice also have increased seizure susceptibility. Despite its clear biomedical importance, the molecular mechanisms by which this G protein regulator operates, and whether it modulates neurotransmission in brain neurons are completely unknown. SYNOPSIS OF AIMS: Based on compelling preliminary data, I propose that the PGR modulates both G?i- and G??-dependent signaling without directly affecting the G protein enzymatic activity (i.e., nucleotide binding and/or hydrolysis), and that this novel mechanism fine tunes GPCR signaling in brain neurons. In AIM#1 I will dissect how the PGR regulates G protein signaling in reconstituted systems (in vitro and cell-based), whereas in AIM#2 I will characterize how it engages physically G?i by combining mass-spectrometry and mutagenesis. In AIM#3 I will characterize how the endogenous PGR regulates neuronal GPCR signaling by using primary cultures of neurons from wild-type and KO mice. Together, the achievement of my goals will lead to the dissection of a previously uncharacterized mechanism of regulation of GPCR signaling with important consequences in normal neural function and neurological disorders such as chronic pain and epilepsy.